In this study a well-characterized pathological mutation at nucleotide position 3243 of human mitochondrial DNA was introduced into human ρ^sup 0^ teratocarcinoma (NT2) cells. In cloned and mixed populations of NT2 cells heteroplasmic for the mutation, mitotic segregation toward increasing levels of mutant mitochondrial DNA always occurred. Rapid segregation was frequently followed by complete loss of mitochondrial DNA. These findings support the idea that pathological mitochondrial DNA mutations are particularly deleterious in specific cell types, which can explain some of the tissue-specific aspects of mitochondrial DNA diseases. Moreover, these findings suggest that mitochondrial DNA depletion may be an important and widespread feature of mitochondrial DNA disease.

MITOCHONDRIAL DNA (mtDNA) mutations are now a well-recognized cause of human disease. Pathological mutations often coexist with apparently wild-type mtDNA, a situation termed heteroplasmy (HOLT et al 1988, 1990; SHOFFNER et al 1990). The mutation at nucleotide position (np) 3243 of human mtDNA was one of the first pathological mutations to be characterized, often associated with the mitochondrial disease, mitochondrial encephalo(myo)pathy, lactic acidosis, and stroke-like episodes, or MELAS (GOTO et al. 1990). In other patients, the same mutation is also linked to diabetes and deafness (MIDD) (VAN DEN OUWELAND et al 1992). The A-to-G transition at np 3243 is located in the transfer RNA gene that decodes leucine UUR codons (GOTO et al. 1990) and also forms part of the binding site for a transcription termination factor (CHRISTIANSON and clayTON 1988). The mutation adversely affects the steady-state level, aminoacylation, and extent of wobble base modification of tRNALeuUUR (CnoMYN et al 1992; KING et al 1992; YASUKAWA et al 2001).

In vitro studies of pathological mtDNA mutations have exploited cell lines that lack mtDNA. These so-called p° cells are fused with cytoplasts from donor cells containing mtDNA mutations. The resultant cybrids manifest the phenotypic consequences of the mutations in control cell backgrounds (CHOMYN et al. 1991). These studies have, in the main, utilized cell lines unrelated to the tissues most affected by mitochondrial disease, such as osteosarcoma and cervical or lung carcinomas. The tissues most commonly affected by pathological mtDNA mutations are, conversely, brain, muscle, cochlea, and pancreatic â-cells.

In an attempt to overcome this methodological problem, we selected, as a recipient for cybridization, a pluripotent teratocarcinoma cell line from which a number of differentiated cell types can be derived (CHADALAVADA et al. 2005), including cells displaying neuronal properties (YouNKiN etal. 1993). We depleted (undifferentiated) NT2 cells of their endogenous mtDNA by prolonged treatment with dideoxycytidine (ddC), thus creating the desired p° line. The same rationale lay behind a study of two mutations associated with Leber's hereditary optic neuropathy in the NT2 cell background (WONG etal 2002).

When mitochondria derived from a MELAS patient with a mixture of wild-type and A3243G mutant mtDNA were introduced into p° NT2 cells, segregation toward increasing levels of mutant mtDNA invariably occurred. …

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